Multi-drug resistant (MDR) bacterial infections represent a significant threat to public health. It is estimated that the impact of antibiotic resistance will continue to grow resulting in a global economic burden of $100 trillion and causing 10 million deaths annually. Many of the therapeutics currently under development represent traditional antibiotic approaches or next generation antibiotics that will likely suffer a limited market lifespan due to the rapid emergence of resistance. To effectively address antibiotic resistance and have therapeutic options that are effective, approaches that utilize new mechanisms of action must be explored. Arrevus is developing a novel approach to addressing MDR bacterial infections using Designer Proline-rich antimicrobial peptide Chaperone protein inhibitors (DPCs), derived from insects and selectively modified, acting as inhibitors to one of the critical bacterial proteins responsible for bacterial protein folding, DnaK. Preliminary studies have demonstrated the potential of DPCs as antibiotic potentiating agents against MDR gram-positive and gram-negative bacterial pathogens. Our efforts have shown that DPCs: 1) enhance antibiotic activity; 2) reduce bacterial burden in systemic infection models; 3) have favorable preliminary safety profiles; and 4) provide an enhancement to antimicrobial agents through a novel mechanism of action. While early efforts to develop DPCs has been successful, the pace of the programs and expansion into additional indications has been hindered due to the lack of reliable in vitro screening assays that correlate with in vivo efficacy. DPCs that have displayed potency using minimum inhibitory assays have failed in subsequent in vivo efficacy studies. The objective of the proposed Phase I program is to develop and validate an in vitro assay for DPC activity. In Aim 1, a scratch test assay using human alveolar and keratinocyte cells will be characterized for several endpoints, including scratch closure, cytokine production, and apoptosis in the presence of well-characterized DPCs and antibiotics to develop an assay that is predictive of in vivo DPC activity. In Aim 2, the assay developed in Aim 1 will then be used to inform a medical chemistry program that is designed to identify DPCs with activity against methicillin-resistant Staphylococcus aureus. Two lead DPCs identified using the using the assay will then be evaluated for in vivo efficacy using a standard thigh infection model. Successful completion of the proposed program will provide proof-of-concept for the use of the developed in vitro assay for detecting DPC activity that is predictive of in vivo activity and will support a Phase II program that will expand the assay to use with gram- negative pathogens and evaluate lead DPCs in indication-specific models.